Project Summary Shortly after birth, most mammalian cardiomyocytes (CMs) become postmitotic and nonregenerative, concurrent with becoming polyploid (either binucleated or mononuclear tetraploid). What controls this process has been unknown. As a result, though, it is generally thought that the adult heart has too little regenerative capacity to appreciably recover after injury. A subpopulation of mononuclear diploid CMs (MNDCMs), thought to be very small in number, persists in the heart through adulthood and is a candidate cell type to support endogenous heart regeneration. Using a wholly new conceptualization, we demonstrated in inbred mice that the percentage of MNDCMs in the adult heart, and the degree of functional recovery and of CM proliferation after adult heart injury, are both highly variable traits subject to the combined influence of multiple polymorphic genes. We identified and confirmed the CM- specific kinase Tnni3k as one such gene with polymorphic alleles that influence variability in the adult level of MNDCMs and thereby in the level of CM regeneration after adult infarction. Using this new understanding, the focus of this project is to elucidate the processes that cause CMs to become polyploid and postmitotic, and the consequences of these processes. In Aim 1, we will dissociate the two roles of Tnni3k in the natural neonatal heart after birth and in the adult heart following injury; we propose that these are related but independent and both relevant to the outcome after adult injury. In Aim 2, we propose to identify two new genes that also influence how CMs remain proliferative or become postmitotic, and to confirm our insights related to the regenerative capacity of MNDCMs. In Aim 3, we explore an unexpected influence of Tnni3k in the proper function of the cardiac conduction system (the electrical system of the heart), and how this role relates to the process of heart regeneration. In Aim 4, we invoke a mechanistic understanding to unify these observations of CM regeneration and conduction. The conceptual significance of this project is transformational: rather than heart injury resulting inexorably in declining heart function as is currently believed, some individuals may have substantial regenerative ability depending on their genetic and cellular composition. Furthermore, these insights might be developed for therapeutics to improve heart regeneration in all patients regardless of their genetic background. And finally, this project might explain why the mammalian heart transitions in most individuals to become mostly postmitotic in the postnatal period, even at the cost of loss of regenerative ability.